Pie HudaPie Huda

A thesis submitted June 25, 2015 for the degree of Doctor of Philosophy and defended October 1, 2015.

The PhD School of Science
Faculty of Science
Niels Bohr Institute, Structural Biophysics X-Ray and Neutron Science, University of Copenhagen

Supervisor:
 Lise Arleth

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Abstract

Studying membrane proterin structure and function using nanodiscs

The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation.

Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins and a lipid bilayer in defined nanometer size, which can act as a stabiliser for membrane proteins. This enables both functional and structural investigation of membrane proteins in a detergent free environment which is closer to the native situation. Understanding the self-assembly of nanodiscs is important for understanding the key mechanisms during reconstitution of membrane proteins in these lipoproteins. In this project the self-assembly of nanodiscs has been structurally characterized with small angle X-ray scattering (SAXS) in a time resolved fashion. This brought knowledge about the structural development as detergent is removed from the solution. This also provided valuable information useful for optimal reconstitution of membrane proteins in nanodiscs. The knowledge was utilized in the reconstitution of proteorhodopsin in nanodiscs where buffer compositions and reconstitution detergents were varied. The different reconstitutions of proteorhodopsin were subsequently analysed by small angle X-ray scattering to evaluate the structural impact of these factors. This shed light on influences that are important to consider in the reconstitution process. In regards to the structure analysis of membrane proteins in nanodiscs it is desirable to acquire structural information supplementary to that obtained through SAXS. Such information can in theory be obtained using small angle neutron scattering (SANS) by using a D2O based buffer system. Unfortunately D2O induced aggregation is a commonly observed problem for biomolecules which also hampered SANS studies in this project. For this reason nanodiscs were systematically analysed at different D2O buffer compositions varying salt, PH etc. to clarify this problem. Unfortunately, a solid solution was not found but an influencing factor identified.

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